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Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-05-23 to 2005-07-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

European Economic Community (EEC), EEC Directive 92-69, Part C: Methods for the determination of ecotoxicity, publication No. L383, December 1992 C.2. "Acute Toxicity for Daphnia"

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

OECD guidelines for testing of chemicals, guideline No. 202 Part 1 " Daphnia sp., Acute immobilisation Test", Adopted April 4, 1984

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

EC Number:

609-858-6

Cas Number:

406-78-0

Molecular formula:

C4H3F7O

IUPAC Name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

Test material form:

liquid

Specific details on test material used for the study:

Identification: ASAHIKLIN AE-3000
Molecular formula: C4H3F7O
Molecular weight: 200.07
CAS Number: 406-78·0
Description: Clear colourless liquid
Batch: 50407070
Purity: 99.985%
Test substance storage: In refrigerator in the dark
Stability under storage conditions: Stable
Expiry date: 28 February 2006
Specific Gravity: 1.47
Stability in water: Not indicated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

During the final test duplicate samples for analysis were taken according to the schedule below.
Sampling: Frequency At ca. 27 hours following the start of dosing, samples were taken from nominally 22 and 221 mg/I to check the actual exposure concentrations. At the start and the end of the exposure period samples were taken from all concentrations and the control.
Volume: 10 ml from the approximate centre of the test vessel.
Storage: Samples taken at the end of the test were stored in a deep-freeze until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding test (static): Preparation for the range-finding test started with test solutions prepared at 10 and 100 mg/I applying 1 or 13 minutes of magnetic stirring, respectively. Care was taken that during preparation as little as possible volatilisation occurred (weighing and preparation in closed vessels which already contained a small volume of test medium, short preparation period}. The lower test concentrations were prepared by dilution of the test solution of 10 mg/I. The final test solutions were all clear and colourless.

First flow-through test: The project was continued with a limit test under flow-through conditions. The nominal test concentration was 221 mg/I, which was expected to correspond to an actual exposure concentration close to 100 mg/I. A blank-control was included. Test procedure and conditions were similar to those applied in the final flow-through test.

Final flow through test: Concentrations:
1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether: Nominal 22, 40, 71, 123 and 221 mg/I, expected to correspond to actual concentrations close to 10, 18, 32, 56 and 1 00 mg/I.

Controls: Test medium without test substance or other additives (blank-control).

Dosing system:
Sufficient amounts of pure test substance were collected daily in airtight sealed vials and used as 'stock' solutions for dosing. The pure test substance was dosed via a computer-controlled system in combination with 5 dispensers (Gilson). Via this system the dosed volumes entered a mixing flask separately from the tap water supply. The tap water was supplied via a flow meter with a flow rate of ca. 12 l/h. In the mixing flask the dosed volumes and the tap water were mixed under continuous stirring. The whole system was checked daily. The hourly dosed volumes in combination with a tap-water flow of 12 liters per hour resulted in a nominal concentration range between 22 and 221 mg/I. However, anticipating the rapid loss of test substance, it was expected that this dosing regime would give an actual concentration range between 10 and 100 mg/I.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the lest selection of young daphnia with an age of <24 hours, from parental daphnids of more than two weeks old.

Breeding:
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22 °C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

18-22 mg/l constant within 2°C

pH:

6.0-8.5, not varying by more than 1 unit

Dissolved oxygen:

>7 mg/l at the start of the test, >=5 mg/l at the end of the test.

Nominal and measured concentrations:

Static Range finding test: 0, 0.1, 1.0, 10 or 100 mg/l (nominal)
First flow-through test: 0 or 100 mg/l (nominal)
Final flow-through test: 10, 18, 32, 56 or 100 mg/l (nominal) - 0, 4.7, 15, 12, 55 or 94 mg/l (geometric mean measured concentration)

Details on test conditions:

Test duration: 48 hours
Test type: Flow-through, continuous renewal of test media.
Test vessels: ca. 1.5 litres, stainless steel containing four stainless steel meshes and airtight closed with a Perspex lid.
Medium: Tap water
Number of daphnia: 20 per concentration
Loading: 5 per mesh
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnia: Two days after the start of the dosing to permit stabilisation of the system.

Measurements and recordings
Immobility (including mortality): At 24 hours and at 48 hours.
pH and dissolved oxygen: At the beginning, after 24 hours and at the end of the test, for all concentrations and the control.
Temperature of medium: Continuously in the vessel containing the blank-control, beginning at the start of the test.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 94 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Static Range finding test: No significant immobilisation of daphnids was observed at any of the concentrations tested. Analytical results of samples taken from 100 mg/I showed that the mean measured
concentrations were stable at approximately 5 mg/I during the 48-hour test period. Hence, with the current preparation procedure actual exposure concentrations close to nominal could not be reached. To enable testing higher actual concentrations and to increase the security of maintaining these stable it was decided to continue testing applying a flow-through system. All further test conditions were maintained within the limits prescribed by the protocol.

First limit flow-through test: A total of 80% of the daphnids exposed to the limit concentration were immobilised after 48 hours of exposure. Analyses of samples taken from nominal 221 mg/I showed that the measured concentration was in agreement with what was anticipated and expected, i.e. 100 mg/I (when expected evaporation is taken into account). It should however be noted that during exposure droplets were present on the bottom of the exposure vessels. This could have had a direct effect on the immobility if the droplets originated from undissolved pure test substance, which was expected based on the test substances specific gravity of 1.47. Consequently, the observed immobility averaging 30% after 24 hours and 80% after 48 hours should be taken with care. Furthermore, relatively high numbers of daphnids were trapped at the surface. Hence, the present test results were influenced by mechanical disturbances and therefore considered not representing the intrinsic toxicity of the test substance. It was consequently decided to continue with a final flow-through study testing a range of concentrations and to pay special attention not to include undissolved test substance droplets in the test vessels by increasing the stirring rate in the mixing vessels. The pH ranged between 7.9 and 8.0, the oxygen concentration was between 8.4 and 8.9 and the temperature was kept between 21.1 and 21.5°C. Hence, all test conditions in the limit test were maintained within the limits prescribed by the protocol.

Final flow-through test:
Measured concentrations: Analyses of the samples taken from nominal 22 and 221 mg/I one day before the start of the test showed mean measured concentrations of 7.7 and 95 mg/I indicating that the system functioned
properly and that the actual concentrations were close to the anticipated targets of 10 and 100 mg/I. All concentrations measured at the start of the experiment were in agreement with what was expected, except at the nominal concentration of 71 mg/l. This concentration was lower than measured at nominal 40 mg/I. Concentrations remained stable at the two highest concentrations, but decreased in the lower concentrations. However, only the highest exposure concentration was essential for determination of the toxicity parameters. The mean concentration measured in the highest test group corresponded to 94 mg/I.

Immobility: Immobility percentages ranged between 5 and 30% in the different concentrations tested. Hence, a 50% immobility rate was not reached in any of the concentrations tested, including a highest tested concentration of 94 mg/I. There was no clear relation between the actual exposure concentrations and the observed immobility. At termination of the study some droplets were observed in the four highest exposure concentrations indicating a possible disturbing effect on the mobility rates due to undissolved test substance. Comparison of the results of the highest exposure concentration with those obtained in the limit test showed that concentrations were comparable but effects were significantly lower in the final test. It can be argued that the higher effects in the limit test were likely to be related to the presence of relatively large amounts of precipitate (droplets) and the high numbers of floaters. Floaters were not observed in the final test.

Experimental conditions: These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: >7 mg/I at the start, >=5 mg/I at the end of the test). The temperature of the test medium was 20.5°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.3 and 20.7°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

CONCLUSION: The 48h-EC50 was >94 mg/I under the conditions of the present flow-through study with Daphnia magna exposed to 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether.

Results with reference substance (positive control):

REFERENCE TEST
Start: 21 June 2005
End : 23 June 2005
48-hour Acute Toxicity Study in Daphnia magna with K2Cr2O7 (NOTOX Project 442531).
The study procedures described in this report were based on the ISO International Standard 6341, the EEC directive 92/69, Part C.2. "Acute toxicity for Daphnia", December 1992, and the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test', Adopted April 4, 1984.

The reference test was carried out to check the sensitivity of the test system as used by NOTOX. Daphnia were exposed for a maximum of 48 hours to K2Cr201 concentrations of 0.10, 0.18, 0.32,
0.56, 1.0 and 1.8 mg/1 and to a blank control. Twenty daphnia were exposed per concentration. The reference substance, potassium dichromate (K2Cr2O7, art. 4864, batch no. K28974764) was
obtained from Merck, Darmstadt, Germany.

The actual responses in this reference test with K2Cr201 were within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of Daphnia magna
was in agreement with the historical data collected at NOTOX.

The 24h-EC50 was 0.84 mg/I with a 95% confidence interval between 0.78 and 0.94 mg/I.

The 48h-EC50 was 0.53 mg/I with a 95% confidence interval between 0.46 and 0.64 mg/I.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48h-EC50 was >94 mg/I under the conditions of the present flow-through study with Daphnia magna exposed to 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether.

Executive summary:

The acute toxicity of 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether to Daphnia magna was determined in a guideline study undertaken according to OECD TG 202. The 48h-EC50 was >94 mg/I under the conditions of the present flow-through study with Daphnia magna exposed to 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether.